Plasmodium vivaxmalaria remains one of the most preva-lent parasitic diseases in tropical regions, mainly on the cen-tral Asian and South American continents[1]. Estimatesof annualP. vivaxcases range from 75 to 90 million. Al-
Abbreviations:rPvMSP-114, rPvMSP-120, recombinant 14 and 20 kDapolypeptides, belonging to the 33 kDaPvMSP-1 cleavage fragment;PfMSP-1,Plasmodium falciparummerozoite surface protein 1; HRBAP,high reticulocyte binding activity peptide; HABPs, high activity bindingpeptides; aa, amino acid; ELISA, enzyme linked immunosorbent assay;IFAT, immunofluorescent antibody test; T-Boc, terbutoxycarbonyl; HPLC,high-pressure liquid chromatography; MS, mass spectrometry; CD, cir-cular dichroism; Ig, immunoglobulin; PBS, phosphate-buffered saline pH7.3; BSA, bovine serum albumin; VCG-1, Vivax Colombia Guaviare-1;PI, day 0 pre-immune; I20, 20 days after the first immunisation; II10, II15
and II20, 10, 15 and 20 days after the second immunisation, respectively∗ Corresponding author. Tel.:+57-1-4815219/3244672x141;
though public health measures for controlling this diseaseare being applied, drug-resistantP. vivax strains continueto spread, in particular, chloroquine-resistant ones. The ge-ographical distribution of CQ-resistantP. vivax has morerecently extended to India, Myanmar, Guyana and Brazil[2]. Further strategies aimed at controlling the disease arestill needed, an anti-malaria vaccine being one of the mostimportant.
P. falciparumMerozoite Surface Protein-1 (PfMSP-1) hasbeen one of the most thoroughly studied proteins in thePlas-modiumspecies. Ten interspecies’ conserved blocks havebeen identified within its sequence, suggesting a conforma-tional or functional restriction[3]. The primary polypeptidechain is cleaved into 82, 30, 38 and 42 kDa fragments[4];the 42 kDa C-terminal fragment is further cleaved into 33and 19 kDa fragments in a calcium-dependent process[5–7].19 kDa polypeptide, containing two domains similar to theepidermal growth factor (EGF), is the only portion of theprotein remaining attached to the newly formed ring stage
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parasite after invasion[8]. Such proteolytic cleavage has notbeen demonstrated inP vivax, even though it is thought thata similar process occurs inPvMSP-1, due to similar proteaserecognition sites[9]. Monoclonal and human antibodies di-rected against the first EGF-like domain within the 19 kDafragment have been found to inhibit in vitroPlasmodiumfalciparuminvasion. Immunisation with the whole moleculePfMSP-1[10], or with its constitutive 42 and 19 kDa frag-ments, recombinantly expressed, have shown partial protec-tion in Aotusmonkeys[10–12]. Moreover, protection withsynthetic peptides derived from this protein has been demon-strated both inAotusmonkeys and humans[13,14].
PvMSP-1, a 200 kDa glycoprotein, has been consideredto be a good candidate for aP. vivax malarial vaccineagainst asexual erythrocyte stages, due to its homology withPfMSP-1. PvMSP-1 42 and 19 kDa fragments’ immuno-genic properties have been observed in mice immunisedwith several recombinant segment-adjuvant combinations[15–18]. Assays evaluating PvMSP-119 recombinant frag-ment protective efficacy, when bound to two tetanus toxinT-helper cell universal epitopes inSaimiri boliviensismon-keys, have shown to provide some protection againstP.vivaxexperimental infection, when homologous Sal-1 strainchallenge was performed[19,20].
Fourteen high reticulocyte binding activity (HRBAP) pep-tide sequences have been identified in previous studies us-ing 88 non-overlapping, 20-mer peptides covering the entirePvMSP-1 amino acid sequence, suggesting this protein’s im-portant role in target cell attachment and parasite invasion[21]. Six of these reticulocyte binding peptides are conservedwhen aligned with both Sal-1 strainPvMSP-1 sequence andseveral isolates sequenced at our laboratory[22].
Two polyhistidine-tagged recombinant polypeptides con-tained in the putative 33 kDa fragment were expressed inE. coli and purified under native conditions using Ni-NTAaffinity chromatography. Previous immunological assayshave revealed that these fragments are recognised byP. vi-vax infected patients’ sera[22]. Aotus nancymaaemonkeyswere immunised with either rPvMSP-114 or rPvMSP-120 recombinant fragments, a mixture of both (rPvMSP-114–rPvMSP-120), and synthetic HABPs (included withinthePvMSP-1 33 kDa fragment). Their humoral immune re-sponse was tested by Western blot, IFAT, ELISA and theirprotective efficacy assessed by experimental challenge withthe heterologousP. vivax VCG-1 strain. These assays mayserve as a pre-clinical evaluation to test these recombinantpolypeptides to be included in a vaccine againstP. vivax.
2. Materials and methods
2.1. Antigen preparation and characterisation
rPvMSP-114 and rPvMSP-120 polypeptides, contained inthePvMSP-133 kDa C-terminal region, and a cocktail of thehigh activity reticulocyte binding peptides present in this
region: (1735 (aa 1339–1358), 1738 (aa 1399–1418) and1747 (aa 1579–1598)), were used for immunisation and im-munological studies. rPvMSP-120 comprises aa 1317–1425and rPvMSP-114 comprises aa 1554–1624. rPvMSP-114and rPvMSP-120 polypeptide antigens were expressed inan E. coli system as previously described[22]. All aa po-sitions are numbered according to Belem strainPvMSP-1sequence. pQE recombinant plasmid clones, encodingrPvMSP-120 and rPvMSP-114 polypeptides, were amplifiedby PCR and each product was sequenced by Sanger’s mod-ified method using an ABI PRISM 310 PE genetic analyserto determine the specific polymorphic variant used for im-munisation. The recombinant polypeptides were purifiedusing a polyhistidine-tagged protein affinity purificationsystem (Ni-NTA) under native conditions. Purified proteinswere separated by SDS-PAGE and electroblotted onto ni-trocellulose. Both proteins were pure when analysed bySEC-Supherose 12 HR 10/30 PBS Mr: 1000–3× 105.
Polymerised HABPs were also used as antigens formonkey immunisation, hereinafter prefixed by p: p1735(EILVPAGISDYDVVYLKPLA aa 1339–1358), p1738(SDLNPFKYSPSGEYIIKDPY aa 1399–1418) and p1747(EVKSSGLLEKLMKSKLIKEN aa 1579–1598). Both theHABPs and HABPs plus one glycine and one cysteineresidue added at their amino and carboxi terminus (forfurther polymerisation) were synthesised by SPPS T-Bocmethodology[23]; MBHA resin was used. The Boc groupwas removed by 55% TFA in DCM; side protection groupsand resin peptides were removed by using HF low/highprocedure. Peptides were lyophilised and then characterisedby MALDI-TOF mass spectroscopy. Both were analysedby CD spectra. To ensure that polymers had a similar struc-tural configuration to monomers. CD measurements wererecorded in a Jasco-J810 spectropolarimeter, between 200and 260 nm, at 0 and 37◦C in PBS, in a 1.0 cm quartz cell.
2.2. Animals, immunisation and boosting
Forty-nine splenectomisedAotus nancymaaemonkeysfrom the Colombian Amazon region and 30 spleen-intactAotus monkeys (negative for evidence of previousPlas-modiuminfection assessed by IFAT at 1:20 dilution usingP. falciparum and P. vivax schizont air dried slides) wereused. Animals were observed daily by trained personnel,weighed weekly and bled biweekly for complete bloodcount and serum collection, and as needed for clinicalchemistry. The animals were under the supervision of abiologist specialised in primatology. All animals used inthis study were treated according to conditions previouslyestablished by the Colombian Ministry of Health[13] andregulated by Law 84, 1989, as well as Office for Protectionfrom Research Risks (OPRRs, Department of Health &Human services, USA) international guidelines for AnimalCare. Each animal was immunised with a 300�l dose ofFreund’s Complete Adjuvant plus 50�g antigen (detailedbelow) and a second 300�l dose on day 20 with a Freund’s
A.Y. Sierra et al. / Vaccine 21 (2003) 4133–4144 4135
Incomplete Adjuvant plus 50�g antigen. Animals weredivided into 10 groups, described as follow.
Groups from 1 to 5 were constituted by splenectomisedmonkeys, and groups from 6 to 10 were non-splenectomisedmonkeys. Group 1 included 10 monkeys immunised withrPvMSP-114 polypeptide; group 2 was formed by 10 mon-keys stimulated with rPvMSP-120 polypeptide; group 3was made up of 12 monkeys vaccinated with rPvMSP-114–rPvMSP-120 polypeptide mixture; in group 4, sixmonkeys were inoculated with HABPs polymers p1735(aa 1339–1358), p1738 (aa 1399–1418) and p1747 (aa1579–1598); group 5 included 11 monkeys immunised withPBS; groups 6–10, were constituted by six monkeys each,and received the same vaccination scheme as groups 1–5,respectively.
2.3. Humoral immune response
2.3.1. Antibody assays (ELISA)Antibody titres were evaluated for each one of the
antigens. Monkeys were sampled on days PI, I20, II10,II 15 and II20. The sera were used to measure antibodypresence against rPvMSP-114 and rPvMSP-120 recombi-nant proteins, rPvMSP-114 and rPvMSP-120 polypeptidecocktail, and each one of the following high bindingpeptides: 1735, 1738 and 1747 as well as those pep-tides which did not bind to reticulocytes: 5575 (NDDDG-EEDQVTTGEAESEAP aa 1319–1338), 1736 (GMYKTIK-KQLENHVNAFNTN aa 1359–1378), 1737 (ITDMLDSR-LKKRNYFLEVLN aa 1379–1398), 1746 (QDYNKMDEK-LEEYKKSEKKN aa 1559–1578) and 18512 (SKEIL-SQLLNVQTQLLTMSSEHT aa 1599–1618).
2.3.2. Western blotBlood samples were taken from aP. vivax-infected mon-
key (with parasitaemia over 6%) and passed through a CF11cellulose column to isolate red blood cells. These werelysed with 0.25% saponin and washed three times with PBSto obtain parasites; they were then lysed with 5% SDSplus a protease inhibitor cocktail. Proteins in the extractwere size-separated by electrophoresis in 7.5–15% gradi-ent SDS-PAGE and then electroblotted onto a nitrocellulosemembrane. The membrane was blocked with 0.5% Tween20 in TBS, 5% skimmed milk and split into rows to be in-dependently assayed against individual sera. Before havingcontact with the membrane, every serum was delipidified,inactivated, pre-adsorbed againstE. coli lysate and incu-bated with each one of the monkey sera (PI, I20, II10, II15and II20) 1:100 diluted in blocking buffer for 1 h at roomtemperature. Hyperimmune monkey sera were used as pos-itive controls; the respective pre-immune autologous serumwas used as negative control. Membranes were then washedfive times with 0.5% Tween 20 in TBE and incubated with a1:500 phosphatase-linked goat anti-AotusIgG. After a 0.5%Tween 20 in TBE wash, a phosphatase Substrate Kit wasused to develop Western blot assays.
2.3.3. Immunofluorescent antibody test (IFAT)Asexual blood-stage parasite antibody titres were deter-
mined by IFAT [24]. Multi-spot slides coated withP. vi-vax infected blood cells were allowed to air dry at roomtemperature for 48 h. Prior to use, slides were blocked for10 min with 1% skimmed milk and incubated for 30 minwith two-fold monkey sera sample dilutions (PI, I20, II10,II 15 and II20), starting at 1:40 dilution. Goat antiAotusIgGFITC-conjugate at 1:100 dilution was added after washingwith PBS. Slides were incubated at 37◦C for another 30 minand washed with PBS. Antibody titres were determined byfluorescence microscopy. Monkey and human patient serahaving more than threeP. vivaxepisodes were used as posi-tive controls; pre-immune autologous sera were used as neg-ative controls.
2.4. Challenge
20 days after the second immunisation each one of theanimals was intravenously challenged with 2.5× 106 of thePlasmodium vivaxVCG-1 strain asexual blood-stage par-asites from previously infectedAotus nancymaaemonkeydonors (manuscript submitted for publication). After day 5monkeys were followed-up daily for the development of par-asitaemia by quantitative Giemsa-stained films and AcridineOrange staining determined by fluorescence microscopy.Monkeys were treated with 30 mg chloroquine administeredover 2 days (15 mg per day) following conclusion of the ex-periment (day 16) or when parasitaemia levels reached≥6%.
2.5. Sequence analysis
The nucleotide sequence encoding each one of the re-combinant polypeptides used for immunisation, and thecorresponding segments of the VCG-1 strain used in thechallenge, were analysed as previously reported[22], todetermine whether an heterologous or an homologous chal-lenge was being performed. Sequences were carried outby Sanger’s modified method in an ABI PRIMS 310 PEgenetic analyser. Results obtained were compared to theBelem and Salvador-1 strains.
3. Results
3.1. Antigen preparation and characterisation
The amino acid sequence for rPvMSP-120 was inferredfrom position 1317 (glycine) to aa 1425 (isoleucine) andfor rPvMSP-114 from position 1554 (threonine) to aa 1623(cysteine) based on the DNA sequencing results (Fig. 1A).The polymorphic positions observed are discussed below.6x-His tagged rPvMSP-114 and rPvMSP-120 purity wasevaluated by size exclusion chromatography on a Supherose12 HR 10/30 Mr (1000–3× 105 Da) column. Molecularweight comparison displayed polypeptides having different
4136 A.Y. Sierra et al. / Vaccine 21 (2003) 4133–4144
Fig. 1. (A) Comparison sequences of (a) Belem, Sal-1 and VCG-1 strains with rPvMSP-120 and (b) Belem, Sal-1 and VCG-1 strains with rPvMSP-114. (B)rPvMSP-114 and rPvMSP-120 analysis by SEC Supherose 12 HR 10/30 PBS Mr: 1000–3× 105. Different polypeptide forms corresponding to monomer,dimer, trimer and tetramer molecular weight are shown. (C) Structural configuration by CD spectra for monomer peptides and their respective polymers.
polymerisation states (monomers, dimers, trimers andtetramers) (Fig. 1B), and each one of them was recognisedby an anti-histidine antibody by Western-blot (data notshown). Similar structural configuration between peptidepolymers and their respective monomers was observed byCD. Peptides exhibited a distorted�-turn structure, accen-tuated in peptides 1747 and p1747 at 0◦C and 30% TFE(Fig. 1C).
3.2. Humoral immune response
3.2.1. Antibody assays (ELISA)Although none of the studied groups’ sera displayed
recognition to any of the tested antigens on days I20 andII10 (data not shown), significant differences were ob-served some days after (II15 and II20). The evaluation ofrPvMSP-114 and rPvMSP-120 immunised groups showed
A.Y. Sierra et al. / Vaccine 21 (2003) 4133–4144 4137
Fig. 2. ELISA. Antibody titres were evaluated for each one of the antigens. Significant recognition is observed for rPvMSP-120 and rPvMSP-114
recombinant polypeptides. Splenectomised monkeys were sampled on days 0, II15 and II20.
detectable antibody levels in splenectomised monkeys ondays II15 and II20 when tested against each recombinantprotein and each one of the HABPs (Fig. 2). The splenec-tomised monkey group immunised with the high bind-ing peptide mixture displayed significant recognition forpeptides 1738 and 1747. Non-binding peptides were notrecognised (data not shown).
3.2.2. Western blotSplenenectomised monkey sera groups immunised with
either rPvMSP-114, rPvMSP-120 or HAPBs mixture wereable to recogniseP. vivaxdenatured antigen when assayed
by Western Blot. 42 and 33 kDa bands, corresponding tothe expectedPvMSP-1 cleavage fragments comprising theimmunogens tested, were recognised on day II15 and II20 inthe evaluated groups. There was no recognition of the twosegments in the control group (group 5) or in pre-immunemonkey sera belonging to the four groups immunised witheither the recombinants or the peptide mixture (Fig. 3).
3.2.3. IFATAs observed inTable 1, sera from all (excepting controls)
splenectomised monkey groups displayed low antibody titresable to recogniseP. vivaxas native antigen in IFAT. Monkey
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Fig. 3. Western blot assays. ImmunisedAotus monkeys sera were tested at 1:100 dilution for the presence of antibodies recognisingP. vivax lysate.(A) Pre-immune sera (groups 1, 2 and 3); (B) 21026 monkey serum (immunised with rPvMSP-114); (C) group 3 sera on day II20 (immunised with therPvMSP-114 and rPvMSP-120 mixture). Molecular weight markers are shown at the left, and the immune sera recognised bands are shown at the righthand side with their respective weight, including PvMSP-1 42 and 33 kDa cleavage fragments, as well as lower molecular weight bands of 30 and 28 kDa.
21026 (Group 1) showed the highest antibody titre (1:640 onday II20) (Fig. 4) but no correlation regarding protection wasobserved (treated on day 14). Only two monkeys out of six,in the spleen intact group 8 immunised with the recombinantpolypeptide mix, developed antibody titres, as assessed byIFAT (Table 1).
Table 1Humoral immune response induced by rPvMSP-120, rPvMSP-114 and HABP peptides in splenectomised and spleen intactAotus nancymaaemonkeys
Group no. Antigen inoculate IFA I20 IFA II 10 IFA II 15 IFA II 20
Data shown in parentheses refers to the lowest positive sera dilution within the particular group.
3.3. Immunisation and challenge
In general, immunised monkeys remained in good healthand only adjuvant-related granulomatose local reactionwas observed in all groups. No post-immunisation orpost-challenge adverse systemic effects were observed.
A.Y. Sierra et al. / Vaccine 21 (2003) 4133–4144 4139
Fig. 4. IFAT. Immunofluorescence microscopy onP. vivaxmature schizontsusing 21026 monkey serum in a 1:640 dilution.
Challenge was carried out 20 days after the second immu-nisation with 2.5 millionP. vivax VCG-1 strain asexualblood stage parasites taken from infected monkey donors.Nine animals (including control group monkeys) died priorto challenge due to causes not related to the immunisationprocess (viral and bacterial infections, diarrhea, etc). Deadmonkeys belonged to groups 2, 4, 5, 6, 7 and 9.
3.4. Protection
3.4.1. Protection in splenectomised monkeysIn group 1 (10 animals), monkey 21013 immunised with
rPvMSP-114, had very low parasitaemia (0.26% maximum)on day 9 post-challenge; monkeys 21224 and 21144 hadpeak parasitaemias of 3.6% on day 9 that decreased lateron to 2.2% until the end of the experiment; while the sevenremaining monkeys in this group behaved similarly to con-trol group, reaching high parasitaemias (6%) on day 11 andwere treated (Fig. 5).
In group 2 (immunised with rPvMSP-120), monkeys21149 and 21184 exhibited very low parasitaemia levels(the highest 0.8 and 1.2%, respectively, on days 5 and 16).Monkeys 21170 and 21226 had lower parasitemia peaks(2.3 and 3.4%) than those in the control group. The fourremaining animals in this group were treated on day tendue to high parasitaemia levels (over 6%).
In group 3, immunised with rPvMSP-114 and rPvMSP-120,parasitaemia in monkey 21003 peaked on day 5 with just0.04% remaining completely negative during the rest of thetrial. Another monkey from this group, 21230 had very lowparasitaemia on day eleven (0.9%), being controlled there-after. Monkeys 20986, 20974, 21140 and 21222 showedparasitaemia levels lower than control group (<2.8% ondays 11, 10, 13 and 11, respectively), while the six remain-
ing monkeys in this group behaved similarly to controlgroup.
In group 4, two monkeys (21031 and 21019), immunisedwith the high binding peptide mixture, had parasitaemiapeaks of 1.4% on day 10 and 3.5% on day 12, respectively,progressively decreasing towards day 16. The remainingmonkeys reached 6% parasitaemia.
Control monkeys immunised with PBS plus Freund’sadjuvant developed systematically increasing parasitaemiasthat reached≥6% between days 9 and 12, therefore, beingtreated (Fig. 5). Our institute policy is that all challengeexperiments must be finished by days 15–16 to avoidanaemia and problems related to monkey stress due to ma-nipulation, blending and handling; all monkeys are thustreated.
3.4.2. Protection in non-splenectomised monkeysIn group 6 (immunised with rPvMSP-114) four monkeys
exhibited very low parasitaemia (peaks 0.28, 0.3, 0.42 and1.10% on days 13, 11, 6 and 11, respectively); the other twodied, due to reasons different to the immunisation protocol(Fig. 6).
In group 7 (immunised with rPvMSP-120) no parasiteswere detected in monkeys 21069 and 21077, whilst 21092and 21096 presented very low parasitaemia (0.26 and 0.24%on days 12 and 13) and monkey 21051 peaked at 3.2% onday 12.
In group 8 (immunised with rPvMSP-114 and rPvMSP-120) monkeys 21057, 21082 and 21097 were free of infectionduring the whole trial, whilst 21070 and 21087 peaked at0.32 and 3.2% on days 8 and 11, respectively.
In group 9 (immunised with the high binding peptide mix-ture) monkeys 21090 and 21102 were treated when para-sitaemia levels reached 6%. In this group, 21063 and 21071had lower parasitaemia levels (1.1 and 0.24% on days 13and 7, respectively).
Control group no. 10 (non-splenectomised, PBS immu-nised control group) controlled their parasitaemia naturally,this being no greater than 0.8%.
3.5. Sequence analysis
Three polymorphic amino acid changes were found whenrPvMSP-120, Sal-1, Belem and VCG-1 sequences werecompared. Belem, Salvador-1 and VCG-1 share a glycineand an alanine at positions 1323 and 1337, respectively;these amino acid positions were replaced in rPvMSP-120,the first by a triptophan, and the second by a glutamicacid. At the third polymorphic position (aa 1408), Sal-1,VCG-1 and rPvMSP-120 shared a serine, that was replacedby a proline in the Belem strain. The rPvMSP-120 polypep-tide amino acid sequence proved to be identical to severalpreviously reported sequences from Colombia, Brazil andSouth Korea (GenBank accession numbers: AAC63104,AAN86238, AAN86240, AAF91168, AAF91170 andAAM22864) [22,25].
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Fig. 5. Parasitaemia curves from the immunised splenectomisedAotus nancymaaemonkey groups (1–5). The parasitaemias were evaluated daily afterchallenge until day 16, both by Giemsa and acridine orange staining. Parasitaemia was reported as the parasitised red blood cell percentage.
A.Y. Sierra et al. / Vaccine 21 (2003) 4133–4144 4141
Fig. 6. Parasitaemia curves from the immunised spleen-intactAotus nancymaaemonkey groups (6–10). The parasitaemias were evaluated daily afterchallenge until day 16, both by Giemsa and acridine orange staining. Parasitaemia was reported as the parasitised red blood cell percentage.
4142 A.Y. Sierra et al. / Vaccine 21 (2003) 4133–4144
No variations were found for the rPvMSP-114 polypeptidewith respect to Sal-1, Belem or VCG-1 strains. When onlythese two segments were analysed, VCG-1 strain sequencewas identical to Sal-1 strain (Fig. 1A).
4. Discussion
In designing a protectiveP. vivax malaria vaccine, theimmune response should be directed towards proteins ex-pressed during the different stages ofP. vivax’s life cycle.These proteins should also be exposed on the parasite’s sur-face and functionally involved in the host cell invasion andselection process.
Since genetic polymorphism in functionally importantproteins is considered one of the mechanisms by whichP.vivaxevades immune system recognition, it is also essentialto select genetically conserved fragments amongst differentisolates.P. falciparum MSP-133 is essentially dimorphic,with considerable sequence diversity between the two al-lelic families[26], butPvMSP-133 (containing rPvMSP-114and rPvMSP-120 recombinant polypeptides) displayed verylow genetic variation. These recombinant polypeptides havebeen shown to be functionally important (due to their prefer-ential reticulocyte binding activity) and naturally antigenic[22], making them good vaccine candidates. This study wasdesigned to evaluate their immunogenic and protective prop-erties in intact and splenectomisedAotus nancymaaemon-keys.
Several methods were tested to demonstrate recombinantprotein immunogenicity. First, induced antibodies were ca-pable of recognising denaturedPvMSP-1 42 and 33 kDaputative cleavage fragments (analogous to those obtainedby proteolythic processing ofPfMSP-1) by Western blot.Secondly, detectable, although low, antibody titres by IFAT,reacted with the parasite in native conditions. Third, reactiv-ity to recombinant segments and HABPs was observed byELISA. These humoral immunogenicity results were foundin both protected and non-protected monkeys, suggestingthat other factors, in addition to antibody titres, could bemediating protection (IgG subclasses, antibodies affinity,cellular immunity, etc.)[27–29]. In view of the factorsmentioned above, further studies are needed to determinethe mechanisms involved in protection.
Protection was observed in animals immunised withrecombinant fragments, when challenged with the heterol-ogousP. vivaxVCG-1 strain. One monkey immunised withrPvMSP-114 was fully protected and two others were ableto control infection, exhibiting very low parasitaemias. Twomonkeys immunised with rPvMSP-120 were fully protectedand two more controlled the infection. Although similaroverall protection was observed when comparing recombi-nant fragment mixture with respect to peptide mixture, fullprotection was only achieved when recombinants were usedas immunogens (either combined or alone), suggesting apossible role in protection of either a structural factor or
a contribution of sequences flanking HABPs (not presentin peptide mixture). Previous immunisation studies withP.falciparum conserved high activity binding synthetic pep-tides have shown that these are generally non-immunogenicand non-protective, acquiring immunogenicity and inducingprotection only when certain residues are substituted[30].Therefore, protection assays using modifiedP. vivaxHABPsare needed, to render them immunogenic and protective.
It was observed that spleen intact control group mon-keys immunised with PBS were able to control the infec-tion when evaluating parasitaemia by acridine orange andGiemsa staining, probably due to the ability of their spleens(as primary immune system organ) to depurate altered RBCsfrom circulation[31]. According to these results found ingroup 10, protection and low parasitaemia results, observedin spleen intact monkeys from groups 6 to 9, are not sig-nificant. On the contrary, splenectomised monkeys immu-nised with PBS, employed as control group, were sensitiveto developing the disease as the inoculated parasite dose(2.5×106) was highly infectious, reaching 6% parasitaemiabetween days 10 and 12 post-challenge. This effect was re-producible since the six monkeys from this group had thesame behaviour. These findings validate the results fromparasitaemia obtained in the four splenectomised monkeygroups, showing also, that intact monkeys do not representa good model for evaluatingP. vivaxpreclinical protectiontests when using VCG-1 challenge.
Changes were found in two amino acids when therPvMSP-120 and rPvMSP-114 immunised fragments wereanalysed and compared with the nucleotide sequences en-coding the corresponding fragments in the VCG-1 strain(with which the challenge was done). Glycine was changedfor tryptophan in aa 1323 and alanine for glutamic acidin aa 1337, leading us to conclude that a heterologouschallenge had taken place (Fig. 1A). These changes werealso found in several previously reported isolates of diverseorigin (Brazil, Colombia and South Korea). Although im-munisation and challenge were done with different strains,there was protection in several monkeys, indicating that thestudied region is a good candidate for a multi-strainP. vivaxvaccine.
In previous studies, immunogenic properties conferredby recombinantPvMSP-142 and PvMSP-119 kDa frag-ments were demonstrated in mice, comparing severaladjuvant-protein combinations[15–18]. Two independentassays, testing protective efficacy usingPvMSP-119, cou-pled to two tetanous toxoid T-helper cell epitopes, showedpartial protection in intactSaimiri boliviensismonkeys, af-ter three 250�g doses, when homologous challenge withSal-1 strain was carried out[19,20].
In the present study, we have shown, not only immuno-genicity, but also full protection in some splenectomisedAotus nancymaaemonkeys after vaccination with just two50�g doses of rPvMSP-114 and rPvMSP-120 mixture, andfurther challenge with 2.5 millionP. vivaxasexual stage par-asites (25 times the amount used in previous studies[19,20]).
A.Y. Sierra et al. / Vaccine 21 (2003) 4133–4144 4143
The highly conserved rPvMSP-114 and rPvMSP-120polypeptide mixture, which had been shown to be func-tionally implicated in reticulocyte recognition and naturallyantigenic in humans in previous reports[22], was testedin preclinical protection assays, showing that it is safe,immunogenic and protective in splenectomisedAotus nan-cymaaemonkeys susceptible to developingP. vivaxmalariawith high parasitaemia levels. Therefore, these recombi-nant polypeptides could be good candidates for inclusionin a subunit, multi-stage, multi-antigenicPlasmodium vivaxvaccine.
Acknowledgements
This research project was supported by the ColombianPresident’s Office, and The Ministry of Public Health. Weare greatly indebted to Jason Garry, Jimena Cortes, PilarMartinez, Diana Tovar, Paula Cardenas, Yago Pico de Coaña,Yolanda Lopez, Gabriela Delgado, Ana Maria Espinosa, Di-ana Zea and Veronika Franco and at our Institute, for theirtechnical assistance, and Professor Manuel E. Patarroyo forhis invaluable comments and suggestions.
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